Reverse transcriptase (RT) inhibition of PCR at low concentrations of template and its implications for quantitative RT-PCR.
Numerous instances of reverse transcriptase (RT) inhibition of the PCR were observed while developing nonquantitative uncoupled RT-PCR techniques for detecting nitrogenase and ammonia monooxygenase gene expression in situ. The inhibitory effect of RT on the PCR was removed with increasing template concentrations beyond 10(5) to 10(6) copies. Including T4 gene 32 protein during the reverse transcription phase of the RT-PCR reaction increased the RT-PCR product yield by as much as 483%; if gene 32 protein was introduced after reverse transcription but prior to the PCR phase, no improvement in product yield was observed. Addition of 1 microgram of exogenous calf thymus DNA or yeast tRNA did little to relieve RT inhibition of the PCR on both genomic DNA and mRNA templates. These results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect. Furthermore, the working hypothesis of RT inhibition below a 10(5) to 10(6) copy threshold has important implications for quantitative RT-PCR studies. In particular, competitive, quantitative RT-PCR systems will consistently underestimate the actual RNA concentration. Hence, enumerations of RNA templates below 10(5) to 10(6) copies will be relative to an internal standard and will not be an absolute measure of RNA abundance in situ.
Chandler
D P
DP
Pacific Northwest National Laboratory, Richland, Washington 99352, USA. dp_chandler@pnl.gov
Wagnon
C A
CA
Bolton
H
H
D004268:DNA-Binding Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D012194:RNA-Directed DNA Polymerase / Q000494:pharmacology*; D019914:Taq Polymerase / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D012313:RNA; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase